Protocol Archive

The authors adapted a previously-described method (Boecker et al., 2020, 2021; Fernandopulle et al., 2018) for differentiating iPSCs stably expressing mNGN2 at a safe-harbor locus into human excitatory glutamatergic neurons. Pre-i 3Neuron iPSCs (human iPSCs with an integrated doxycycline-inducible mNGN2 transgene in the AAVS1 safe-harbor locus) were a gift from M. Ward (National Institutes of Health, Maryland).

This protocol generates human medium spiny neurons (MSNs) from induced human pluripotent stem cells (iPSCs).

A method to identify potential interactors of any Green Fluorescent Protein (GFP) tagged protein expressed in mammalian cells by GFP immunoprecipitation coupled to Tandem Mass Tag (TMT) mass spectrometry analysis. The authors used a GFP-tagged phosphoRab interactor protein (RILPL1-GFP), and its non-binding mutant (RILPL1 -GFP, which cannot interact with phosphorylated Rab proteins) as a control.

This protocol can be adapted to isolate organelles from commonly cultured cells, such as HEK293 and A549 cells, that express an organelle tag.

The organelles purified using this method are highly enriched, intact, largely contaminant-free and can be used for various downstream applications, including immunoblotting analysis and proteomic analysis (as described in dx.doi.org/10.17504/protocols.io.ewov1o627lr2/v1), but also lipidomic or metabolomic analysis (as described in dx.doi.org/10.17504/protocols.io.bybjpskn). 

This protocol details about immunoprecipitation using anti-HA magnetic beads.

This protocol details how to isolate nuclei from frozen brain tissue for single nuclear RNA sequencing using 10x Genomics GEM isolation using the Chromium accessory and Single Cell 3ʹ Reagent Kits.

Collection of protocols of Team Deleidi used in the publication: "LRRK2 kinase activity regulates GCase level and enzymatic activity differently depending on cell type in Parkinson’s disease."

This protocol details standard operating procedure (SOP) for systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in non-human primates.

Protocol used to create LRRK1 RCKW grids for cryo-EM used in Snead, Matyszewski, Dickey et al.

The method uses the fluorescent compound anthranilic acid (2-AA) to label oligosaccharides prior to analysis using normal-phase high-performance liquid chromatography. The labeling procedure is rapid, selective, and easy to perform. With the inclusion of a 2AA-labeled chitotriose calibration standard, it is possible to obtain accurate and reproducible molar quantities of individual GSL species.

This protocol details the cryosectioning of the mouse brain.  

This protocol details about plasmid-reprogramming of human fibroblasts.

Associated with the following preprint (published September 30th 2021): Progranulin deficiency results in reduced bis(monoacylglycero)phosphate (BMP) levels and gangliosidosis. bioRxiv 2021.09.30.461806; doi: https://doi.org/10.1101/2021.09.30.461806

Here, authors describe an approach for purification of early/sorting endosomes, providing a means by which to examine early aspects of the endolysosomal system (Endo-IP) and to combine this with lysosome purification using Lyso-IP. This allows one to examine the proteome, lipidome, as well as electron microscopy imaging of endosomes.

Here the authors present an immunofluorescence protocol to assess the extent of colocalization between FLAG-EEA1 and RAB5 with and without the Dynamin-1 and -2 (DNM1/2) inhibitor Dyngo4a.

Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide (GlcCer), a membrane glyco-sphingolipid, to ceramide and glucose. This assay detects GBA activity by using a fluorogenic substrate that reacts with cell lysates previously treated with or without CBE (GBA1 inhibitor).

The objective of this protocol is to provide directive on how to extract lipids from plasma, cells, tissue, and purified organelles for analysis by liquid chromatography (LC)-MS. This will typically yield quantitative data for more than 200 lipids, depending on the sample type analyzed, across a range of lipid classes: phospholipids, cardiolipins, sphingolipids, di- and triacyclglycerols, and cholesterylesters.

This protocol describes the standard procedure of using collagenase to passage human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs).

Protein purification protocol for tag-less LRRK2RCKW as done by Leschziner and Reck-Peterson Labs. Same protocol can be used to purify LRRK1RCKW as well. Original protocol by David Snead. Modified by Yu Xuan Lin and Mariusz Matyszewski for publication.